BIOCATALYSIS
liquids for improved stability of the lipase in the presence of the acidic chemocatalysts and for reduction of zeolite-catalysed side reactions.30,31 The substrates and supercritical carbon dioxide were fed continuously, thus omitting the use of organic solvents for product extraction. A reaction sequence for the continuous synthesis of D-xylulose from D,L-serine and D,L-glyceraldehyde involving three enzymes in a single continuously stirred tank reactor (CSTR) was presented by Bongs.32 Here, a D-amino acid oxidase was used to form hydroxypyruvic acid from D-serine, which was subsequently converted to D-xylulose by a transketolase-catalysed reaction with added D,L-glyceraldehyde. Catalase was used to regenerate the reduced cofactor FADH by oxidation with oxygen. In order to decrease enzyme deactivation caused by shear forces, bubble-free aeration through a silicon membrane was implemented. A space-time yield of 4.3 g/l per day was achieved. However, further optimisation of the process was not feasible due to the complexity of the system caused by possible cross-inhibition, deactivation, and reactant instabilities. All of the single-reactor continuous (chemo)enzymatic processes mentioned, except for the one described by Bongs,32
employed a maximum of two different catalysts. To the best of this article’s authors’ knowledge, until now there is no established continuous (chemo)enzymatic process catalysed by four or more isolated catalysts combined in one pot. Therefore, it seems that a simple single-reactor or ‘in-pot’ approach would hardly cope with more complex multi- catalyst systems. Even in the three-enzyme process, Bongs already encountered problems preventing further optimisation of the process. There are three reasons for this: (a) Different enzymes usually work best under different, sometimes even incompatible, reaction conditions, eg pH, temperature, ionic strength, or the presence of metal ions; (b) intermediates or mediators of certain steps can inhibit enzymes catalysing other steps (cross-inhibition); (c) certain enzymes may act on the reactants involved in other steps, and thus catalyse undesirable side reactions. Nevertheless, it may be assumed that in the future continuous multi-step chemoenzymatic processes involving more than three catalysts will be realised in a single reactor as well. For instance, Liu et al developed a packed-bed reactor containing seven enzymes co-immobilised through hexahistidine tags on nickel-agarose beads (‘super beads’).33 These enzymes catalyse a complex network of sequential and coupled reactions, in which galactose, uridine monophosphate (UMP) and inorganic polyphosphate are converted to uridine
diphospate galactose (UDP-Gal) in the presence of catalytic amounts of ATP and glucose 1-phosphate. When the reaction mixture containing starting materials and cofactors was circulated for 48 hours through the reactor, 50% of UMP was transformed to UDP-Gal. Although in their work the authors did not establish the continuous production of UDP-Gal, in principle such a reactor could be operated in continuous mode as well, if the immobilised enzymes were more active and stable enough.
REFERENCES For a complete list of references contact the authors.
This is the first part of a three-part article based on a review by Ruslan Yuryev, Simon Strompen and Andreas Liese of the Institute of Technical Biocatalysis, Hamburg University of Technology and published in Beilstein J. Org. Chem. 2011, 7, 1449–1467.
The complete review article is part of the Thematic Series ‘Chemistry in flow systems II’ edited by A. Kirschning © 2011 Yuryev et al; licensee Beilstein-Institut.
Further information Andreas Liese Institute of Technical Biocatalysis Hamburg University of Technology 21073 Hamburg Germany Email:
liese@tuhh.de
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